Protocol to measure centromeric array size changes using droplet digital PCR-based quantification of higher-order repeats

Summary Centromere length changes occurring during somatic cell divisions can be estimated by quantifying the copy numbers (CNs) of higher-order repeats (HORs), which are nested repeats of monomers that comprise centromeric arrays. Here, we present a protocol for single-cell isolation for clonal evolution followed by droplet digital PCR-based quantification. The assay measures HOR CNs across subclones to determine the frequency and degree of changes in HOR CNs. This protocol tests the underlying molecular mechanisms responsible for rapid centromere sequence evolution. For complete details on the use and execution of this protocol, please refer to Showman et al.1

c. Incubate the cells at 37 C for 2-3 days until 80% confluence.d.Remove old (conditioned) media from the flask and place in a 50 mL conical tube.e. Wash cells in the flask once with 1X PBS.f.Trypsinize cells in 3 mL of 0.25% trypsin-EDTA and incubate at 37 C for 5 min.g.Add 7 mL of media and break up all clumps of cells into individual cells by pipetting up and down.
Optional: Although conditioned media may be used, avoid using it from cells that are overly confluent due to the potential presence of excess cellular waste.
2. Isolate single cells from the population of U2OS cells by limiting dilution. 5,6. Measure the cell number of the homogenized cells with a Vi-cell analyzer.b.Add 10 6 cells/mL in a 1.5 mL microcentrifuge tube (refer to Preparation of HOR CN control).c. Dlute the cells to 10 5 cells/mL in a 1.5 mL microcentrifuge tube containing media and homogenize by pipetting.d.Measure the cell number of the 10 5 cells/mL stock.e. Dilute the 10 5 cells/mL stock solution in 1:10 (10,000 cells/mL) and 1:100 (1,000 cell/mL) using 1.5 mL microcentrifuge tubes containing media.f.Filter the conditioned media through a 0.22 mm PES filter.g.Seed 50 single cells in a 96-well plate by adding 50 mL of the 1:100 diluted cell solution in 10.5 mL of conditioned media and mix thoroughly by pipetting.h. Mve the cell-containing media to a reservoir and dispense 100 mL of cell solution into each well of a 96-well plate except for one well on the right bottom corner of the plate (H12).This well will be used to focus the microscope while searching for single cells.i. Add 100 mL of the 1:10 cell solution in the bottom right corner well. jKeep the plate undisturbed in an incubator for 7 days.
CRITICAL: It is essential to homogenize the cell solution during dilution in order to yield a high number of single cells with minimal doublets.
Optional: A hemocytometer or other cell counter may be used instead of a Vi-cell analyzer.
3. Scan the 96-well plate to find monoclonal cells on the 7 th day post isolation.
a. Focus the microscope using the cells in the bottom right corner well (H12).b.Scan the entire plate and mark the wells containing single colonies.c.Keep monitoring the marked wells every day at roughly the same time and discard any well containing more than a single colony or that are observed to grow at a different rate than the majority.
CRITICAL: Having two cells in a well reduces assay sensitivity due to genetic heterogeneity.
Optional: We tested a microfluidic-based single cell sorter, yet the limiting dilution was as efficient as the single cell sorter and has an easier and less expensive workflow.

Note:
We observed the plate at the same time every day from day 7 th to 14 th after single cell isolation to ensure that all selected clones are monoclonal and growing at the same rate.
4. Expand the monoclonal colonies on the 14 th day post isolation.a. Wash the wells that contain monoclonal colonies twice with 1X PBS.b.Trypsinize the monoclonal colonies in 100 mL of 0.25% trypsin-EDTA and incubate at 37 C for 5 min.c.Add 100 mL of media and break up all cell clumps into individual cells by pipetting up and down.d.Transfer 200 mL of the cell-containing media to a single well in a 12-well plate.e. Add 800 mL of media to each well containing cells in a 12-well plate.
Note: We noticed that some colonies are not easily detached during trypsinization from a 96-well plate.To minimize cell loss, make sure to check cells under a microscope to determine whether they are fully detached.

5.
Once the monoclonal colonies in the 12-well plate are confluent, collect the cells.
a. Wash the cells once with 1X PBS.b.Trypsinize in 500 mL of 0.25% trypsin-EDTA (enough to cover the cells) and incubate at 37 C for 5 min.c.Add 500 mL of media (R volume of trypsin added) and break up all cell clumps into individual cells by pipetting up and down.
Cryopreservation of monoclonal cell lines 6. Select a few monoclonal cell lines from above and expand the cells by placing each individual monoclonal cell line in a separate T-25 flask.7. Add 5 mL of media and incubate at 37 C until the cells are confluent.8. Freeze the cells at 1.5 3 10 6 cells per cryovial in 1 mL of media containing 5% DMSO.9. Store the cells at À80 C.

CRITICAL:
The purpose of this step is to preserve monoclonal colonies that are genetically homogeneous.Pre-existing genetic heterogeneity will lead to an overestimation in array change.The frozen stocks will be used to initiate clonal evolution.
Preparation of HOR CN control 10.Centrifuge the remaining parent cells after single-cell isolation at 300 3 g for 5 min.11.Remove the supernatant.12. Store the parent cell pellet at À20 C until the droplet digital PCR (ddPCR) run is needed.

Preparation of positive control
13. Thaw a CHM13 cryostock containing 2 3 10 6 cells and expand the cells to 10 7 cells.CRITICAL: Vortex all reagents and spin down before preparing the reaction.When preparing the ddPCR reaction mixture, add 1.25 x the total required volume because the viscosity of 2X supermix may lead to a pipetting error (see Figure 2 Column 6).Prepare a reaction in multiples of 8 because the automatic droplet generator takes 8 samples (1 column of a 96-well plate) and generates droplets simultaneously.

STEP-BY-STEP METHOD DETAILS
Isolation of subclones from a monoclonal cell line for clonal evolution c.Prepare a working solution for the samples and two standards.Each standard and unknown sample requires 190 mL of working solution.Add 1.9 mL of 100X dye in the 188.1 mL buffer per measurement.Scale up as needed to make enough volume to use.d.Vortex to mix thoroughly.e. Aliquot 190 mL of the working solution in a thin, UV-transparent 0.5 mL tube for each sample and standard.f.Add 10 mL of standard and unknown sample to the assay tubes.g.Vortex tubes to mix.h.Incubate the samples and standards at room temperature for 5 min.i. Generate a standard curve by DeNovix DS-11 FX using two standards.j.Measure the concentration of the samples.
Optional: any assay that can quantify 100 pg-250 ng range fluorometrically including Qubit dsDNA assays can be used.
24. Vortex thoroughly and dilute samples to 2 ng/mL in Buffer AE using Rainin low retention tips.25.Measure the concentration of 2 ng/mL stock using a DeNovix dsDNA ultra-high sensitivity assay.
a. Equilibrate all components to room temperature before use.b.Vortex and spin down all components.c.Prepare a working solution for the samples and two standards.Each standard and unknown sample requires 200 mL of working solution.Add 0.5 mL of the 400X dye and 2 mL of the 100X enhancer in 197.5 mL buffer per measurement.Scale up as needed to make enough volume to use.d.Vortex to mix thoroughly.e. Aliquot 200 mL of the working solution in a thin, UV-transparent 0.5 mL tube for each sample and standard.f.Add 10 mL of standard and unknown sample to the assay tubes.g.Vortex tubes to mix.h.Incubate samples and standards at room temperature for 5 min.i. Generate a standard curve by DeNovix DS-11 FX using two standards.j.Measure the concentration of the samples.26.Repeat steps 24-25 until 2 ng/mL (< G 10%) stocks are made from all samples.27.Vortex thoroughly and dilute 2 ng/mL stock to 1:20 in Buffer AE using Rainin low retention tips.

CRITICAL:
The CN of the reference single gene is used to normalize the CN of the HOR of the same subclone.To minimize subsampling error, it is important to make the 2 ng/mL stock as accurately as possible and mix thoroughly before preparing the diluted gDNA stock.
Pause point: Diluted gDNA can be stored at À20 C.

ddPCR reaction setup
Timing: 1 h (96 samples) CRITICAL: We measure the HOR and reference single gene (SG) CNs in separate reactions using the EvaGreen assay due to constraints caused by the differences in ddPCR dynamic range between the HOR and reference SG when using the same amount of gDNA input.Therefore, we use 2 ng of gDNA for the reference SG measurement and a 1:20 dilution of 2 ng gDNA stock for HOR measurement.This is the optimal condition to quantify D11Z1 CN in U2OS cells.The optimal gDNA concentration for each different SG and dilution factor for each different HOR should be determined based on the CN of target HOR and SG in the cell of interest to fit within the dynamic range of the ddPCR.CRITICAL: EvaGreen dye binds to both double-stranded DNA and nonspecifically singlestranded DNA.Therefore, concentrations of gDNA input and primer need to be optimized to ensure accurate CN measurement.CRITICAL: To minimize subsampling error, use low retention tubes and tips for all steps and pre-wet tips to avoid bubbles in the tips.CRITICAL: Avoid introducing air bubbles to the droplet generator cartridge.This will impact droplet generation and reduce the assay efficiency.Therefore, add 10% excess volume (22 mL) to prevent air bubble formation (see Figure 2. Column 4).
28. Thaw the diluted gDNA stocks of subclones, parental cells, and ddPCR control.29.Vortex thoroughly to homogenize gDNA in solution.30.Place 2X supermix and 20X primer pair stocks at room temperature to thaw.31.Following the ddPCR reaction mix table in the materials and equipment section above, prepare two ddPCR master reaction mixes without gDNA (the gDNA will be added in step 35) according to the number of samples needed, in triplicate (see Figure 2. Column 5) and including the two controls.
Note: One master mix will include the HOR target primers and the other will include the single gene reference primers.
Note: Refer to Figure 1 for a suggested plate layout and Figure 2 for an example of the master mix volume calculation.
CRITICAL: Incubate the ddPCR reaction with the restriction enzyme for at least 30 min to ensure every CN of the HOR and SG are isolated and partitioned into droplets.
32. Label 1.5 mL low retention microcentrifuge tube that match the number of samples that are being measured.33.Vortex thoroughly and aliquot 69 mL of reaction master mix to each tube.34.Vortex thoroughly to mix the gDNA.35.Add 3.6 mL of either 2 ng/mL (SG), 1:20 dilution of the 2 ng/mL stock (HOR), or ddH 2 O to the corresponding tubes containing the target primer-pair.36.Vortex thoroughly to mix the reaction.37. Incubate the reaction in the dark for 30 min.38.Vortex tubes thoroughly to homogenize the reactions.39.Aliquot 22 mL from each tube to each corresponding well in a 96-well ddPCR plate in triplicate (see Figure 1).40.Turn on the PCR plate sealer which is set to seal at 180 C for 5 s.41.Place a pierceable foil seal on the top of the plate (the redline should be on the top of the plate) and seal the plate.42.Vortex the plate at 2000 rpm for 1 min.43.Centrifuge the plate at 1000 3 g for 1 min.
CRITICAL: Remove air bubbles and ensure the ddPCR reaction mix is at the bottom of the wells.If there are any bubbles after centrifugation, repeat step 42.

Droplet generation
Timing: $45 min (for 96 samples).It will take less time for fewer samples.It takes an average of 3.5 min per column 44.Set up the configuration on the automated droplet generator (AutoDG) as follows.
a. Select oil type as EvaGreen.b.Specify the columns that contain the samples.45.Replace the oil and consumables as follows (the green lights on the instrument decks or touchscreen will turn on if the consumables are placed correctly).a. Ensure that the oil volume is sufficient and the type of droplet generation oil is EvaGreen.b.Load droplet generator cartridges onto the cartridge blocks on the back row (the green gaskets should be on the right side).c.Insert an empty pipet tip waste bin.d.Remove the lids and place pipet tips on the pipet tip blocks on the middle row.e. Place a 96-well ice cold chill block on the droplet plate holder (front right).f.Place a new ddPCR 96-well plate in the 96-well chill block.46.Insert the sealed ddPCR reaction plate into the sample plate holder (front left).47.Start the AutoDG when all requirements are satisfied.48.Once the droplet generation is complete, place a pierceable foil seal onto the 96-well plate containing the droplets in the chill block (the red line should be on the top of the plate).49.Gently remove the plate and seal at 180 C for 5 s.

CRITICAL:
The droplets are very fragile.We recommend processing the PCR amplification step as soon as the droplet generation is complete.Do not vortex or spin down the plate.Handle the plate very carefully until it is loaded on the thermal cycler.CRITICAL: Improper sealing of the plate leads to oil evaporation during thermal cycling and can compromise droplet data quality.
Note: See the AutoDG instrument manual for details: https://www.bio-rad.com/webroot/web/pdf/lsr/literature/10043138.pdfNote: A QX200 droplet generator can be used instead of the AutoDG.See the instrument manual for details: https://www.bio-rad.com/webroot/web/pdf/lsr/literature/10031907.pdfNote: Droplets will have an opaque layer at the top of each well if the droplets are properly generated.

Note:
The chill block needs to be stored at À20 C upside down for more than 2 h before use.The instrument is not sensitive enough to detect errors occurring in this block such as a misplaced sample plate or a failure to place a new plate.A mistake in this step can lead to the failure of the entire run.

Note:
The completion time for droplet generation can vary between runs.We recommend monitoring the instrument performance until the first column is processed before leaving the machine.

Thermal cycling (PCR)
Timing: 2.5 h 50.Transfer the sealed droplet plate to a thermal cycler and start the PCR cycling using the program as below (Table 2).The volume of the thermal cycling reaction is 40 mL.The ramp rate should be 2 C/s for all cycles.Additional master mix should be prepared to prevent bubble formation (1.1x the amount used for a single reaction), to account for triplicate measurement (3x), and Supermix viscosity (1.1x the triplicate volume).The gDNA template (red) will be added separately.The target volume for each sample in triplicate at least 69 mL (blue).See also Figure 1 and Table 1.

CRITICAL:
The annealing temperature must be optimized for every set of primers using a temperature gradient (65 C-55 C).
Note: We noticed that incubating the plate at 4 C overnight after PCR cycling increases the number of accepted droplets.
Pause point: The plate can be stored at 4 C overnight after completion of the PCR step until droplet reading.

Droplet reading
Timing: 2 h (for 96 samples).Droplet reading takes about 10 min for each column 51.Remove the metal retainer and insert the sealed PCR-amplified reaction plate into the QX200 droplet reader and secure the retainer.52.Open the Quantasoft software and prime the QX200 droplet reader.53.Set up a new plate layout according to the experimental samples.54.Designate the parameters as follows.
Note: ddPCR droplet reading will not proceed if the droplet reading oil is insufficient for the reaction or the waste container is full.

EXPECTED OUTCOMES
Successful completion of the monoclonal isolation step should result in greater than 25 monoclonal cells, with an average of 4 doublets, when approximately 0.5 cells/well was added to the 96-well plate.A successful ddPCR run should result in >16,000 accepted droplets and a separation of two peaks representing the amplitudes of the negative (background) and the positive (target).The CN of D11Z1 in the positive control (CHM13) should be $3,500 copies/mL with minimal background in the negative control.

QUANTIFICATION AND STATISTICAL ANALYSIS Overview
The purpose of this section is to quantify the absolute number of copies/mL for both HOR and SG and normalize the HOR CN by SG CN to estimate an average of HOR CN per centromeric array.The HOR CN per array across the subclones will be compared to the value of parental monoclonal cells that

Protocol
the subclones were isolated from.The frequency of CN change across the subclones is determined using an ANOVA test followed by Tukey's Honestly Significant Difference test.The two steps that are needed are as follows.
Use Quantasoft to estimate the CN.
Quantify HOR CN per array by normalizing with the SG CN.
The number of accepted droplets and negative droplets are used to calculate # copies/mL.The number of positive droplets influences the technical error of the measurement.

Data analysis
Timing: 0. Note: For a more conservative approach for defining positive droplets, set the horizontal gate close to the main cluster of positive droplets.

LIMITATIONS
Due to single-cell isolation requirement, this protocol requires four weeks to collect the subclones for measurement.Therefore, the protocol is limited to cell lines that can grow after single cell isolation.A lower number of cell divisions may increase the sensitivity of the CN change detection due to sharing higher genetic homogeneity in monoclonal populations.However, 5 3 10 5 cells were required for gDNA extraction to yield consistent results with less technical error.Since we used previously published and tested primers, 1 no primer design step is included in this protocol.

Figure 1 .
Figure 1.Example plate layout for quantifying copy numbers of higher order repeats (HOR) and reference single gene (SG) across subclones in a 96-well plate In columns 1-6 the HOR measurements are highlighted blue and in columns 7-12 the reference SG are highlighted purple.The parental cells (PC) that provide the baseline HOR and SG CN are highlighted green and orange respectively.The assay positive control is highlighted yellow.All measurements are in triplicate except the assay positive control (yellow) and the no-template control (gray).

Figure 2 .
Figure 2.An example volume calculation for the reaction master mixAdditional master mix should be prepared to prevent bubble formation (1.1x the amount used for a single reaction), to account for triplicate measurement (3x), and Supermix viscosity (1.1x the triplicate volume).The gDNA template (red) will be added separately.The target volume for each sample in triplicate at least 69 mL (blue).See also Figure1and Table1.

5 h 1 .
After completion of the droplet reading, open Quantasoft and select the run.2. Select the well that contains the NTC and ensure the well is free of contamination (see Figure3and Troubleshooting 2). 3. Determine the amplitude threshold to separate the positive and negative droplets for the purpose of quantification as follows.a. Select all experimental wells targeting HORs.b.Click the 1-D amplitude plot view.c.Manually place a horizontal gate to separate the clusters of positive and negative droplets.d.Repeat steps a-c with all wells targeting SG. e. Export the results.csvfile from Quantasoft.

4 .
To further increase data quality, open the results.csvfile and exclude any well that meets the following criteria.a. < 10,000 accepted droplets.b.A distinctively different average amplitude from the wells that share the same target.c.Number of positive droplets are < 100.d.Number of negative droplets are < 10. 5. Multiply the HOR CN x 20 to account for the dilution factor difference between the HOR and SG gDNA input (1:20).6. Normalize the HOR CN with the SG CN by pairing the first HOR and SG replicates and dividing the HOR CN by the SG CN. 7. Repeat step 6 for any other technical replicates.8. Conduct the ANOVA test followed by Tukey's HSD test between each subclone CN and the parental CN value in R.
14. Add 10 6 cells in each 1.5 mL microcentrifuge tube.15.Centrifuge the cells at 200 3 g for 5 min.16.Remove the supernatant.17.Store the cell pellet at À20 C until the ddPCR run is needed.
EMD Millipore Steriflip sterile disposable vacuum filter units (0.22 mm) Fisher Scientific CAT#SCGP00525 Pipette tips RT LTS 200 mL Rainin CAT#30389240 (Continued on next page)MATERIALS AND EQUIPMENTThis protocol uses the QX200 Droplet Digital PCR system which includes an automated droplet generator, PX1 PCR plate sealer, thermal cycler with 96-deep well reaction module, and QX200 droplet reader which comes with QuantaSoft Software (version 1.7).QuantaSoft is compatible with Windows 10 operating systems.The Droplet Digital PCR Applications Guide is available from the Bio-Rad website: https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6407.pdf.CRITICAL: The fresh master reaction mix without the DNA template should be made just before the run.The gDNA of the samples will be added individually.

Table 2 .
PCR cycling conditions